The zebrafish (Danio rerio) is a powerful genetic model to study many features of vertebrate biology in vivo. However, transplantation methods have been lagging in the zebrafish due to lack of knowledge about the zebrafish Major Histocompatibility Complex (MHC) genes. In order to apply the genetic and screening advantages of the zebrafish model to questions involving all aspects of transplantation biology, the functional MHC genes in the zebrafish must be identified. Unlike mice and humans where both Class I and Class II MHC genes are linked to a single chromosomal locus inherited as a haplotype from each parent, there appear to be at least three chromosomal loci in the zebrafish with putative MHC genes by sequence homology. Despite substantial work mapping these MHC genes, there are almost no data characterizing their function. It is unclear which genes are actually expressed on the cell surface, playing a functional role presenting peptide antigens to recognize foreign tissues. We propose to map the functional MHC genes in the zebrafish that are important for immune matching in transplantation. This will be addressed in Specific Aim 1 using a candidate gene approach. Putative zebrafish MHC genes will be individually cloned into an overexpression vector. Zebrafish T cell leukemias will be generated by co-injecting the MHC expression vector and the murine c-Myc oncogene into single-cell clonal CG1 fish. These leukemias will be transplanted into CG1 recipient fish. If the putative MHC gene is functional, the tumor will be rejected. If the MHC gene is not functional, the leukemia will engraft, causing the recipient's death. Specific Aim 2 will use an unbiased approach to map the genes required for rejection of a transplanted tumor. In this case tumors will be generated by injection of the murine c-Myc oncogene into single-cell progeny from serial crosses of clonal CG1 fish and AB/CG1 hybrid fish. Each subsequent generation will have fewer AB MHC alleles, and eventually some progeny will carry only CG1 MHC alleles. These MHC-matched tumors will engraft into CG1 recipients while mismatched tumors are rejected. Standard positional cloning methods will be used to map the relevant gene in tumors that do not engraft compared with tumors from sibling fish that do engraft. Together these aims will identify the functional zebrafish MHC genes, opening the door to numerous transplantation experiments that have previously not been possible. Such experiments would harness the advantages of the zebrafish model, and hence address questions that are difficult or impossible to ask in mammals. Because thousands of sibling progeny can be generated in a few weeks from a single mating pair of zebrafish, large scale chemical and genetic screens related to hematopoietic and tumor transplantation could be performed. In addition, immunology experiments studying antigen presentation, activation of lymphocytes and natural killer cells, and infectious disease models in the zebrafish would become possible. Discovery of the functional MHC genes would open the door for zebrafish researchers studying many aspects of transplantation and vertebrate biology.